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1.
Nat Commun ; 14(1): 4567, 2023 07 29.
Article in English | MEDLINE | ID: mdl-37516778

ABSTRACT

In many bacteria, chromosome segregation requires the association of ParB to the parS-containing centromeric region to form the partition complex. However, the structure and formation of this complex have been unclear. Recently, studies have revealed that CTP binding enables ParB dimers to slide along DNA and condense the centromeric region through the formation of DNA bridges. Using semi-flexible polymer simulations, we demonstrate that these properties can explain partition complex formation. Transient ParB bridges organize DNA into globular states or hairpins and helical structures, depending on bridge lifetime, while separate simulations show that ParB sliding reproduces the multi-peaked binding profile observed in Caulobacter crescentus. Combining sliding and bridging into a unified model, we find that short-lived ParB bridges do not impede sliding and can reproduce both the binding profile and condensation of the nucleoprotein complex. Overall, our model elucidates the mechanism of partition complex formation and predicts its fine structure.


Subject(s)
Caulobacter crescentus , Ear Auricle , Centromere , Chromosome Segregation , Polymers
2.
PLoS Comput Biol ; 17(12): e1009756, 2021 12.
Article in English | MEDLINE | ID: mdl-34965245

ABSTRACT

The spatial localisation of proteins is critical for most cellular function. In bacteria, this is typically achieved through capture by established landmark proteins. However, this requires that the protein is diffusive on the appropriate timescale. It is therefore unknown how the localisation of effectively immobile proteins is achieved. Here, we investigate the localisation to the division site of the slowly diffusing lipoprotein Pal, which anchors the outer membrane to the cell wall of Gram-negative bacteria. While the proton motive force-linked TolQRAB system is known to be required for this repositioning, the underlying mechanism is unresolved, especially given the very low mobility of Pal. We present a quantitative, mathematical model for Pal relocalisation in which dissociation of TolB-Pal complexes, powered by the proton motive force across the inner membrane, leads to the net transport of Pal along the outer membrane and its deposition at the division septum. We fit the model to experimental measurements of protein mobility and successfully test its predictions experimentally against mutant phenotypes. Our model not only explains a key aspect of cell division in Gram-negative bacteria, but also presents a physical mechanism for the transport of low-mobility proteins that may be applicable to multi-membrane organelles, such as mitochondria and chloroplasts.


Subject(s)
Bacterial Outer Membrane Proteins , Escherichia coli Proteins , Intracellular Space , Lipoproteins , Peptidoglycan , Periplasmic Proteins , Protein Transport/physiology , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Cell Division , Cell Wall/chemistry , Cell Wall/metabolism , Escherichia coli/chemistry , Escherichia coli/cytology , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Intracellular Space/chemistry , Intracellular Space/metabolism , Lipoproteins/chemistry , Lipoproteins/metabolism , Peptidoglycan/chemistry , Peptidoglycan/metabolism , Periplasmic Proteins/chemistry , Periplasmic Proteins/metabolism , Protein Binding/physiology
3.
Mol Cell ; 81(19): 3992-4007.e10, 2021 10 07.
Article in English | MEDLINE | ID: mdl-34562373

ABSTRACT

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.


Subject(s)
Bacterial Proteins/metabolism , Chromosome Segregation , Chromosomes, Bacterial , Cytidine Triphosphate/metabolism , DNA, Bacterial/metabolism , Myxococcus xanthus/enzymology , Bacterial Proteins/genetics , Binding Sites , Catalytic Domain , Crystallography, X-Ray , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Hydrolysis , Mutation , Myxococcus xanthus/genetics , Protein Conformation , Structure-Activity Relationship , Substrate Specificity , Time Factors
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